Neuritogenic and neuronal survival promoting peptides derived from the family of s-100 proteins

ABSTRACT

The present invention relates to peptide fragments derived from proteins of the S-100 family promoting neural cell survival, differentiation and proliferation. The invention further relates to pharmaceutical compositions comprising said peptide fragments and uses thereof for treatment of diseases and conditions where the effects of stimulating neural cell proliferation, differentiation and/or survival, and/or stimulating neural plasticity associated with learning and memory are beneficial for treatment.

FIELD OF INVENTION

The present invention relates to neural cell survival differentiationand proliferation promoting peptide fragments derived from proteins ofthe S-100 family, pharmaceutical compositions comprising said peptidefragments and uses thereof for treatment of diseases and conditionswhere the effects of stimulating neural cell proliferation,differentiation and/or survival, and/or stimulating neural plasticityassociated with learning and memory are beneficial for treatment.

BACKGROUND OF INVENTION

The S100 family is a group of vertebrate-specific Ca2+-binding proteinswith a highly conserved primary structure possessing both intra- andextracellular functions. Most S100 family members, including S100A4, areantiparallelly packed homodimers stabilized by disulphide bridges(Donato, 2001). Intracellularly, S100 proteins are involved in a varietyof processes including regulation of cytoskeletal dynamics, Ca2+homeostasis, and cell proliferation and differentiation. Importantly,some S100 proteins can also be secreted, form oligomers owing to thenonreducing conditions of the environment, and exert their effectsacting at the cell surface (Kerkhoff et al., 1998). A plasma membranetarget for S100B and S100A12, the receptor for advanced glycation endproducts (RAGE), has been identified on inflammatory and neural cells(Hofmann et al., 1999). However, RAGE is probably not the sole receptorfor members of the S100 family, since effects of extracellular S100A12and S100B proteins can be observed in cells lacking RAGE (Robinson etal., 2002), and some of these effects are RAGE-independent in cellsexpressing the receptor (Sorci et al., 2002).

The gene of S100A4 (also termed Mts1) was isolated from tumor cells(Ebralidze et al., 1989), where its expression increased the ability ofthe tumor to metastasise. S100A4 has also been detected in normaltissues, in particular, in the nervous system. Both in brain and spinalcord, S100A4 expression appears in astrocytes shortly after the start ofmyelination, with the highest level observed in the areas, in whichneurogenesis takes place, and in regions possessing high plasticity inthe adult (Åberg and Kozlova, 2000). Moreover, in the peripheral nervoussystem, expression of S100A4 increases after sciatic nerve or dorsalroot injury (Kozlova and Lukanidin, 1999). Thus, the release of S100A4from S100A4-positive astrocytes as a result of either secretion or celldamage might play a role in neuronal plasticity under normal andpathological conditions. Importance of S100A4 in brain developmentand/or regeneration is supported by the fact that the protein is apotent promoter of neurite outgrowth in hippocampal neurons in vitro(Novitskaya et al., 2000). Moreover, S100A4 acts as a neuroprotectantfor primary neurons induced to undergo cell death (Pedersen et al.,2004). The molecular mechanism of this effect, including a receptortransducing S100A4 signals, has not been identified. However, theS100A4-induced neurite outgrowth could be reduced by inhibitors ofintracellular Ca2+ homeostasis (Novitskaya et al., 2000). This indicatesthat extracellular S100A4 might affect the intracellular Ca2+concentration ([Ca2+]i) thereby modulating neuronal differentiation.

The crystal structure of human EF-hand calcium-binding protein S100A12in its calcium-bound form has been determined to 1.95 A resolution bymolecular replacement using the structure of the S100B protein (Moroz etal., 2001). Like the majority of S100 proteins, S100A12 is a dimer, withthe interface between the two subunits being composed mostly ofhydrophobic residues. The fold of S100A12 is similar to the other knowncrystal and solution structures of S100 proteins, except for the linkerregion between the two EF-hand motifs. Sequence and structure comparisonbetween members of the S100 family suggests that the target-bindingregion in S100A12 is formed by the linker region and C-terminal residuesof one subunit and the N-terminal residues of another subunit of thedimer. The N-terminal region of the target-binding site includes twoglutamates that are conserved in most of the S100 sequences. The preciserole of S100A12 in cell behaviour is yet undefined, as is the case forthe whole family, although it has been shown that the interaction ofS100A12 with the RAGE receptor is implicated in inflammatory response(Hofmann et al., 1999). Human recombinant S100A12 has been found todramatically induce neuritogenesis of hippocampal neurons (Mikkelsen etal., 2001). The response to S100A12 was dependent on the dose in abell-shaped manner. A 10-fold increase in neurite outgrowth was observedupon treatment with S100A12 in concentrations between 0.1 and 2.0 μmalready after 24 h. Exposure to S100A12 for only 15 min was enough toinduce neuritogenesis when measured after 24 h, but to obtain a maximalresponse, S100A12 had to be present in the culture for at least 4 h. Theresponse to S100A12 is abolished by inhibitors of phospholipase C (PLC),protein kinase C (PKC), Ca²⁺ flux, Ca²⁺/calmodulin dependent kinase II(CaMKII) or mitogen-activated protein kinase (MEK).

REFERENCES

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SUMMARY OF THE INVENTION

The present invention relates to peptide sequences capable ofstimulating neuronal cell differentiation, neuronal cell survival andneural plasticity associated with learning and memory and capable ofinhibiting of inflammatory response. According to the invention, thepeptide sequences described herein comprise a common structural motifwhich confers on the peptides biological activity.

Thus, in the first aspect the present invention relates to a peptidecomprising a sequence of at most 30 contiguous amino acid residuescomprising an amino acid motif of the formula:

x^(p)-(x1)-(x2)-(x3)-x^(p)-(x4),

-   -   wherein    -   x^(P) is a hydrophobic amino acid residue    -   at least one of (x1), (x2) or (x3) is an amino acid residue        selected from E, D, K, R, Q, N, S or T and    -   (x4) is an amino acid residue selected from E, D, K, R, Q, N, S        or T or a hydrophobic amino acid residue.

In another aspect the invention relates to a compound comprising apeptide sequence comprising the amino acid motif of above.

A peptide sequence of the invention is preferably derived for a proteinof the S100 family. The invention discloses particular examples of suchpeptide sequences. These sequences comprise the motif disclosed hereinand are capable of stimulating neuronal cell differentiation, neuronalcell survival and/or neural plasticity associated with learning andmemory.

The invention also relates to a pharmaceutical composition comprising apeptide sequence of the invention and/or compound comprising thereof.

Further aspects of the invention include a method of stimulating neuralcell survival, differentiation, proliferation and/or plasticityassociated with memory and learning comprising using a peptide sequenceaccording to the invention and/or a pharmaceutical compositioncomprising thereof.

Then, the invention relates to uses of disclosed peptide sequences andcompounds comprising thereof for the manufacture of a medicament and/orfor the production of an antibody.

The antibodies of the invention are capable binding to an epitopecomprising the structural motif described herein or a peptide sequencedisclosed herein. The invention also relates to a pharmaceuticalcomposition comprising an antibody of the invention.

The invention also concerns methods of treatment of individuals in needcomprising administering a peptide sequence, compound or antibody or apharmaceutical composition comprising thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Design of S100-derived peptides. The sequences of S100A4 andS100A12 proteins were divided into six regions each, and the tetramericpeptides representing individual regions were generated. The peptideswere termed hekatones (H) 1 to 6. Thereafter, all twelve peptides weretested for their neuritogenic activity in primary neurons.

FIG. 2: Identification of the neurite inducing regions in sequences ofS100A4 and S100A12. The S100A4- and S100A12-derived peptides (H1-H6, seeFIG. 1 for details) were tested for their neuritogenic activity inprimary hippocampal neurons (upper panel, HN) and cerebellar neurons(lower panel, CN). Two neuritogenic regions have been identified in thesequence of S100A4 corresponding to the H3 and H6 peptides, and oneregion has been determined in the S100A12 sequence corresponding to theH3 peptide. The H3 and H6 peptides derived from S100A4 were furthercharacterized in vitro, and are henceforth referred to as H3 and H6.*p<0.05, **p<0.01, ***p<0.001 when compared to the untreated controls.

FIG. 3: Identification of aminoacids responsible for the neuritogeniceffect of the H3 and H6 peptides (upper and lower panels, respectively).First, truncated versions of the peptides were tested for their abilityto induce neurite outgrowth from primary hippocampal neurons.Non-truncated versions were used as controls. It was found that only theC-terminal part of the H3 peptide was crucial for the induction ofneurite outgrowth (upper panel, left), whereas for the H6 peptides,truncation of just two aminoacids from the N-terminal resulted in theloss of neuritogenic activity (lower panel, left). Based on truncationexperiments, a minimal sequence required for the induction of neuriteoutgrowth was identified for each peptide (shown underlined on top ofrespective truncation graphs). These sequences were subsequently usedfor the alascan experiments, where individual amino acids weresubstituted by alanins in order to identify residues crucial for theneuritogenic activities of H3 and H6 (upper and lower panels,respectively, right). We have found four amino acids in each peptidesequence, which were important for the induction of neurite outgrowth byH3 and H6 (shown underlined on top of respective alascan graphs).(*)-statistical significance P<0.05, (**)-p<0.01, (***)-p<0.001, Anovaanalysis.

FIG. 4: H3 and H6 activate intracellular messengers mediating neuriteoutgrowth and survival of primary neurons. Primary hippocampal neuronscultured for 6 hours were stimulated with H3 or H6 for 15 minutes.Thereafter, an activity (phosphorylation level) of the threeintracellular messengers involved in neurite outgrowth and survival,Akt, CREB, and ERK, was determined using the PAGE method. Both peptidessignificantly increased phosphorylation of all three messengers.(*)-statistical significance P<0.05, (**)—P<0.01, (***)—P<0.001, Anovaanalysis.

FIG. 5: S100A4-derived peptides (H3, H6) increase neuronal survival.Primary cerebellar neurons cultured for 7 days in high potassium medium([KCl]=40 mM) were induced to undergo apoptosis by lowering of KClconcentration to 5 mM. Both H3 and H6 peptides partially rescuedcerebellar neurons increasing the survival by 30-40%. (*)-statisticalsignificance P<0.05, (**)—P<0.01, (***)—P<0.001, Anova analysis.

FIG. 6: H6, but not H3 increases intracellular Ca²⁺ concentration([Ca²+]_(i)). Since Ca²⁺ has been demonstrated to play a crucial role inthe processes of neurite outgrowth and survival, and S100A4 is known toincrease [Ca²⁺]_(i) in primary neurons, we loaded cultured hippocampalneurons with a ratiometric Ca²⁺-sensitive dye fura-2 and tested whetherthe S100A4-derived peptides affected [Ca²⁺]_(i). It has been found thatapplication of H6, but not H3 significantly increased the level ofintracellular Ca²⁺. (*)-statistical significance P<0.05, (**)—P<0.01,(***)—P<0.001, Anova analysis.

FIG. 7: H3 and H6 protect the brain from the kainic acid (KA)-inducedtoxicity. Both H3 and H6 decreased amount of seizures induced by 20 mg(kg KA (top). Moreover, the peptides shifted the profile of seizuresinduced by 30 mg/kg KA towards less severe seizures types and decreasedthe mortality by 30-50% (bottom). (*)-statistical significance P<0.05,(**)—P<0.01, (***)—P<0.001, Anova analysis.

FIG. 8: The H3 peptide accelerates functional recovery after sciaticnerve crush in rats. Wistar rats have been subjected to a sciatic nervecrush at day 0. Thereafter, rats were divided into three groups, eachgroup receiving daily injections of either vehicle (Veh), or the H3 andH6 peptides at a dose of 10 mg/kg for 18 days. Every second day, theSciatic Function Index (SFI) was evaluated in all groups based on theanalysis of walking tracks of individual animals (SFI=−100 correspondingto a complete limb paralysis, SFI=0 corresponding to a normal limbfunction). It has been found that the H3 peptide strongly promoted therecovery of the motor function of the injured limb. (*)-statisticalsignificance p<0.05, (**)—P<0.01, (***)—P<0.001, Anova analysis.

FIG. 9: Effect of peptides derived from the S100A4 proteins onDNA-fragmentation at post-lesion day 4. The peptides were administeredsubcutaneously in a dose of 10 mg/kg at day (−)1, 1 and 2. Tunel stainedcells are indicated by arrows.

FIG. 10: Quantification of the effect of peptides derived from theS100A4 proteins on DNA-fragmentation at post-lesion day 4. The peptideswere administered subcutaneously in a dose of 10 mg/kg at day (−)1, 1and 2. *p<0.05, **p<0.01 when compared to the vehicle treated control.

FIG. 11: Effect of peptides derived from the S100A4 protein on thenumber of GFAP-positive cells at post-lesion day 4. The peptides wereadministered subcutaneously in a dose of 10 mg/kg at day (−)1, 1 and 2.**p<0.01 when compared to the vehicle-treated controls.

FIG. 12: Effect of peptides derived from the S100A4 protein on the areaof GFAP-immunoreactivity (% of the total penumbra area). The peptideswere administered subcutaneously in a dose of 10 mg/kg at day (−)1, 1and 2. ***p<0,001 when compared to the vehicle-treated controls.

FIG. 13: Effect of peptides derived from the S100A4 protein on thenumber of proliferating cells at post-lesion day 4. The peptides wereadministered subcutaneously in a dose of 10 mg/kg at day (−)1.1 and 2.*p<0.05 when compared to the vehicle-treated controls.

DETAILED DESCRIPTION OF THE INVENTION 1 Peptide Sequences IndividualPeptide Sequences

First aspect of the invention relates to a peptide comprising at most 30contiguous amino acid residues comprising an amino acid motif of theformula:

x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4),

-   -   wherein    -   x^(p1) and x^(p2) are hydrophobic amino acid residues,    -   at least one of (x1), (x2) or (x3) is an amino acid residue        selected from E, D, K, R, Q, N, S or T and    -   (x4) is an amino acid residue selected from E, D, K, R, Q, N, S        or T or a hydrophobic amino acid residue.

Hydrophobic amino acid residues x^(p1) and x^(p2) of the motif may beselected from any hydrophobic residues, however in some embodimentsresidues L, V, I, M, F or A may be preferred.

Amino acid residues (x1), (x2) and (x3) may be any amino acid residueswith the proviso that at least one of (x1), (x2) or (x3) is an aminoacid residue selected from E, D, K, R, Q, N, S or T. In some embodimentsit may be preferred that any two of (x1), (x2) or (x3) are residuesselected from E, D, K, R, Q, N, S or T. In some other embodiments it maybe preferred that any one of (x1), (x2) or (x3) residues is an aminoacid residue selected from E, D, K, R, Q, N, S or T, any another of(x1), (x2) or (x3) residues is a hydrophobic amino acid residue and thethird residue is any amino acid residue.

In one preferred embodiment of the invention the motif of abovecomprises at least two contiguous hydrophobic residues. The hydrophobicresidues may be any hydrophobic residues selected from A, F, I, L, M, P,V or W. In some further preferred embodiments the two contiguoushydrophobic residues may be preceded or followed by a hydrophilicresidue selected from E, D, K, R, Q, N, S or T. In other furtherpreferred embodiments the two contiguous hydrophobic residues may bepreceded or followed by a hydrophobic residue. The contiguoushydrophobic residues may be in one embodiment the residues x^(p2) and(x4), in another embodiment the residues (x3) and x^(p2), in stillanother embodiments the residues x^(p1) and (x1) or residues (x1) and(x2).

In one preferred embodiment the sequence (x3)-x^(p2)-(x4) of the motifcomprises (x3) which is an amino acid residue selected from E, D, K, R,Q, N, S or T and (x4) which is a hydrophobic amino acid residue. Thelatter hydrophobic residue in one embodiment may be preferably selectedfrom residues P or A, in another embodiment preferable selected fromresidues I or L, and in still another embodiment preferably selectedfrom residues F or M. In another preferred embodiment (x4) may be anamino acid residue selected from E, D, K, R, Q, N, S or T and (x3) isany hydrophobic amino acid residue. The hydrophobic residue (x3) in thiscase may be selected from V, L, I, A or F.

A peptide sequence comprising a motif according to invention maycomprise from 6 to 50 contiguous amino acid residues.

In one embodiment, the sequence may be of about 50 amino acid residuesin length, such as for example from 40 to 49 amino acid resides. Inanother embodiment the sequence may comprise less then 40 amino acidresidues, for example from 30 to 39 amino acid residues, such as lessthen 30 amino acid residues, for example from 20 to 29, such as about 25amino acid residues. The sequence may also be less then 20 amino acidresidues, such as from 10 to 19 amino acid residues, for example from 10to 15 amino acid residues, for example such as 10, 11, 12, 13, 14 or 15amino acid residues, or for example from 16 to 19 amino acid residues,such as 16, 17, 18 or 19 amino acid residues. Peptides comprising lessthen 10 amino acid residues are also in the scope, such as for example6, 7, 8 or 9 amino acid residues. In particular, the invention concernsthe peptides which length is in the range of 6 to 15 amino acid residuesor in the range of 10 to 25 amino acid residues.

A peptide comprising the above described motif may, in particular,comprise a sequence selected from the amino acid sequences identifiedherein as SEQ ID NOs: 1-85 or consists of any of the sequences SEQ IDNOs:1-85, such as for example SEQ ID NOs:1-5. A sequence selected fromSEQ ID NOs:1-5 is a preferred peptide sequence of the invention.

In another preferred embodiment a peptide of the invention may compriseor consist of a sequence selected from SEQ ID NOs:51-68.

In still another preferred embodiment the peptide may comprise orconsists of a sequence selected from SEQ ID NOs:6-22.

In yet another preferred embodiment the peptide may comprise or consistof a sequence selected from SEQ ID NOs:23-42.

In further preferred embodiments the peptide may comprise or consists ofa sequence selected from SEQ ID NOs:43-50, or a sequence selected fromSEQ ID NOs:52-68, or a sequence selected from SEQ ID NOs:69-85.

In more preferred embodiments the invention concern the sequences of SEQID NOs: 1-5.

In one preferred embodiment the peptide may comprise or consists ofsequence NEFFEGFPDKQPRKK (SEQ ID NO: 1), or comprise a fragment orvariant of sequence NEFFEGFPDKQPRKK (SEQ ID NO:1). In another preferredembodiment, the peptide may comprise or and consists of sequenceKELLTRELPSFLGKRT (SEQ ID NO:2), or comprise a fragment or variant ofsaid sequence, in still another preferred embodiment it may comprise orconsists of sequence KQLLTKELANTIKNIK (SEQ ID NO: 3), or comprise afragment or variant thereof. Still, in another preferred embodiments thepeptide may comprise or and consists of sequence DEAAFQKLMSNLD (SEQ IDNO: 4) or sequence CPLEKALDVMVSTF (SEQ ID NO:5), or comprise a fragmentor variant of any of these sequences.

In the present content the fragment is defined as an amino acid sequencecomprising at least 5 contiguous amino acid residues of a sequenceselected from SEQ ID NOs:1-85, such as SEQ ID NOs:1-5, said amino acidsequences comprising two contiguous hydrophobic preceding or following aresidue selected from K, R, D, E, N, Q, S or T.

The variant in the present content is defined as an amino acid sequenceof 6 to 50 contiguous amino acid residues, which has at least 50%sequence similarity with a sequence selected from the sequences of SEQID NOs:1-85, preferably, more then 50% sequence similarity with asequence selected from sequences of SEQ ID NOs:1-85, such as for examplefrom 51% to 60%, preferably more then 60%, for example between 61% and70%, more preferably more then 70% sequence similarity, such as from 71%to 80%-85%, more preferred sequence similarity about 90%, such as from85% to 90%, and even more preferred sequence similarity about 99%. Inanother embodiment, a variant of a sequence selected from SEQ IDNOs:1-85 may be a sequence of SEQ ID NOs:1-85 which comprisesmodifications of amino acid residues discussed below. Other variants ofpeptide sequences of the invention concerned are also discussed below.

Sequence similarity/homology/identity may be may be calculated usingwell known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75,BLOSUM 80, BLOSUM 85, or BLOSUM 90. The terms “sequence similarity”sequence identity” and “sequence homology” are used in the presentapplication interchangeably when referred to a number or percentage ofidentical or similar amino acid residues in two collated amino acidsequences. “Similar amino acid residues” are amino acid residues derivedfrom the same group of “conservative” amino acid residues. The lattergroups are discussed further in the application.

Both, the fragments and variants as above are according to the inventionfunctional equivalents of peptide sequences of SEQ ID NOs:1-85.

In the present application the standard one-letter code for amino acidresidues is applied as well as the standard three-letter code.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide of the invention exists as the free carboxylicacid, this may also be specified as “—OH”. However, the C-terminal aminoacid of a compound of the invention may be the amidated derivative,which is indicated as “—NH₂”. Where nothing else is stated theN-terminal amino acid of a polypeptide comprise a free amino-group, thismay also be specified as “H—”.

Where nothing else is specified amino acid can be selected from anyamino acid, whether naturally occurring or not, such as alfa aminoacids, beta amino acids, and/or gamma amino acids. Accordingly, thegroup comprises but are not limited to: Ala, Val, Leu, Ile, Pro, Phe,Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, HisAib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Also, according to the invention modifications of the compounds/peptidesmay be performed, such as for example glycosylation and/or acetylationof the amino acids. Basic amino acid residues are according to inventionrepresented by the residues of amino acids Arg, Lys, and His, acidicamino acid residues—by the residues of amino acids Glu and Asp. Basicand acidic amino acid residues constitute a group of charged amino acidresidues. The group of hydrophobic amino acid residues is represented bythe residues of amino acids Leu, Ile, Val, Phe, Trp, Tyr, Met, Ala andPro.

The invention relates to naturally occurring, synthetically/recombinantprepared peptide sequence/fragments, and/or peptide sequence/fragmentsprepared by means of enzymatic/chemical cleavage of a biggerpolypeptide, wherein said peptide sequence/fragments are integral partsof said bigger polypeptides. The invention relates to isolatedindividual peptide sequences.

As mentioned above, the invention also relates to variants of peptidesequences described above.

In one aspect the term “variant of a peptide sequence” means that thepeptides may be modified, for example by substitution of one or more ofthe amino acid residues. Both L-amino acids and D-amino acids may beused. Other modification may comprise derivatives such as esters,sugars, etc. Examples are methyl and acetyl esters.

In another aspect “variants” may be understood as exhibiting amino acidsequences gradually differing from the preferred predetermined sequence,as the number and scope of insertions, deletions and substitutionsincluding conservative substitutions increase. This difference ismeasured as a reduction in homology between the predetermined sequenceand the variant.

In still another aspect, variants of the peptide fragments according tothe invention may comprise, within the same variant, or fragmentsthereof or among different variants, or fragments thereof, at least onesubstitution, such as a plurality of substitutions introducedindependently of one another. Variants of the complex, or fragmentsthereof may thus comprise conservative substitutions independently ofone another, wherein at least one glycine (Gly) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Ala, Val, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone alanine (Ala) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Val, Leu, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one valine (Val) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Gly, Ala, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone leucine (Leu) of said variant, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Ala, Val, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one isoleucine (Ile) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Val andLeu, and independently thereof, variants, or fragments thereof whereinat least one aspartic acids (Asp) of said variant, or fragments thereofis substituted with an amino acid selected from the group of amino acidsconsisting of Glu, Asn, and Gln, and independently thereof, variants, orfragments thereof, wherein at least one aspargine (Asn) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Gln,and independently thereof, variants, or fragments thereof, wherein atleast one glutamine (Gln) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Asn, and wherein at least one phenylalanine(Phe) of said variants, or fragments thereof is substituted with anamino acid selected from the group of amino acids consisting of Tyr,Trp, His, Pro, and preferably selected from the group of amino acidsconsisting of Tyr and Trp, and independently thereof, variants, orfragments thereof, wherein at least one tyrosine (Tyr) of said variants,or fragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Trp, His, Pro, preferably anamino acid selected from the group of amino acids consisting of Phe andTrp, and independently thereof, variants, or fragments thereof, whereinat least one arginine (Arg) of said fragment is substituted with anamino acid selected from the group of amino acids consisting of Lys andHis, and independently thereof, variants, or fragments thereof, whereinat least one lysine (Lys) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Arg and His, and independently thereof, variants, orfragments thereof, and independently thereof, variants, or fragmentsthereof, and wherein at least one proline (Pro) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Tyr, Trp, and His, andindependently thereof, variants, or fragments thereof, wherein at leastone cysteine (Cys) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofAsp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

It thus follows from the above that the same functional equivalent of apeptide fragment, or fragment of said functional equivalent may comprisemore than one conservative amino acid substitution from more than onegroup of conservative amino acids as defined herein above. The term“conservative amino acid substitution” is used synonymously herein withthe term “homologous amino acid substitution”.

The groups of conservative amino acids are as the following:

P, A, G (neutral, weakly hydrophobic),S, T (neutral, hydrophilic)Q, N (hydrophilic, acid amine)E, D (hydrophilic, acidic)H, K, R (hydrophilic, basic)L, I, V, M, F, Y, W (hydrophobic, aromatic)C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide of the invention or fragment thereof. Itmay however also be desirable to introduce non-conservativesubstitutions, particularly, but not limited to, a non-conservativesubstitution in any one or more positions.

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of the peptide of the invention wouldfor example differ substantially in polarity, for example a residue witha non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)substituted for a residue with a polar side chain such as Gly, Ser, Thr,Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, orLys, or substituting a charged or a polar residue for a non-polar one;and/or ii) differ substantially in its effect on peptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like. Exemplary amino acid substitutions which takevarious of the foregoing characteristics into consideration are wellknown to those of skill in the art and include: arginine and lysine;glutamate and aspartate; serine and threonine; glutamine and asparagine;and valine, leucine and isoleucine.

As it was mentioned above the present invention relates to fragments andvariants of the peptide sequences described above. The followingfragments and variants are preferred by the invention.

A fragment which is a sequence which is about 40%, more preferably atleast 50%, more preferably at least 60%, more preferably at least 70%,more preferably at least 80%, more preferably at least 90%, morepreferably at least 95% of the length of a sequence selected from thesequences of SEQ ID NOs: 1-85. It may be preferred a fragment thatcomprises at least 6 amino acid residues and the motifx^(p)-(x1)-(x2)(x3)-x^(p)-(x4), wherein x^(p) is a hydrophobic aminoacid residue, at least one of (x1), (x2) or (x3) is an amino acidresidue selected from E, D, K, R, Q, N, S or T, and (x4) is an aminoacid residue selected from E, D, K, R, Q, N, S or T or a hydrophobicamino acid residue. It may also preferred a fragment which comprises atleast 5 contiguous amino acid residues of a sequence of SEQ ID NOs:1-85,comprising two contiguous hydrophobic amino acid residues preceded orfollowed by a residues selected from E, D, K, R, Q, N, S or T.

A variant which is an amino acid sequence having at least 60%, morepreferably at least 70%, more preferably at least 80%, more preferablyat least 90%, more preferably 95% homology to a sequence comprising themotif of the invention, in particular to a sequence selected from thesequences of SEQ ID NOs: 1-85, or is an amino acid sequence having atleast 60%, more preferably at least 70%, more preferably at least 80%,more preferably at least 90%, more preferably 95% positive amino acidmatches compared to a sequence comprising the motif of the invention, inparticular to a sequence of SEQ ID NOS: 1-85. A positive amino acidmatch is defined herein as an identity or similarity defined by physicaland/or chemical properties of the amino acids having the same positionin two compared sequences. Preferred positive amino acid matches of thepresent invention are K to R, E to D, L to M, Q to E, I to V, I to L, Ato S, Y to W, K to Q, S to T, N to S and Q to R. The homology of oneamino acid sequence with another amino acid is defined as a percentageof identical amino acids in the two collated sequences. The homology ofthe sequences may be calculated using well known algorithms such asBLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;

The preferred fragments and variants of the invention functionalhomologues/equivalents of sequences identified as SEQ ID NOs:1-85, whichmeans that the fragments and variants has at least some biologicalactivity of the original sequence, for example a capability ofstimulating neural plasticity, such as associated with neural celldifferentiation and/or such as associated with memory and learning,stimulating of cell survival, such as inhibiting apoptosis, and/oractivating a receptor.

As mentioned above, the invention relates both to naturally occurring,synthetically or recombinantly prepared peptides and peptides preparedby means of enzymatic/chemical cleavage of a bigger polypeptidesequence. The peptides produced by enzymatic cleavage of a biggerpolypeptide sequence, as well as peptides, which are prepared by meansof recombinant expression or by means of chemical synthesis, whereinsaid peptide sequences are corresponding to integral sequences of biggerpolypeptides or proteins, are according to invention derived from thesequences of said bigger polypeptides or proteins.

The invention preferably relates to peptides which are derived from thesequences of the proteins belonging to the S100 family of proteins, inparticular, S100A1 (Swiss-prot Ass. number: P23297), S100A2 (Swiss-protAss. number: P29034), S100A3 (Swiss-prot Ass. number: P33764), S100A4(Swiss-prot Ass. number: P26447), S100A5 (Swiss-prot Ass. number:P33763), S100A6 (Swiss-prot Ass. number: P06703), S100A7 (Swiss-protAss. number: P31151), S100A8 (Swiss-prot Ass. number: P05109), S100A9(Swiss-prot Ass. number: P06702), S100A10 (Swiss-prot Ass. number:P60903), S100A11 (Swiss-prot Ass. number: P31949), S100A12 (Swiss-protAss. number: P80511), S100A13 (Swiss-prot Ass. number: Q99584), S100A14(Swiss-prot Ass. number: Q9HCY8), S100A16 (Swiss-prot Ass. number:Q96FQ6), S100B (Swiss-prot Ass. number: P04271), S100G (Swiss-prot Ass.number: P29377), S100P (Swiss-prot Ass. number: P25815) and S100Z(Swiss-prot Ass. number: Q8WXG8).

In one preferred embodiment a peptide may consists of or comprise asequence corresponding to a fragment of sequence of S100A4. Such peptideaccording to the invention is derived from S100A4 protein. Examples ofinteresting peptides which are derived from S100A4 protein representedin the application by SEQ ID NOs: 4, 23-42 and 81.

2. Compound

A compound may contain a single copy of an individual amino acidsequence selected from any of the described above, or it may contain twoor more copies of such amino acid sequence. This means that compound ofthe invention may be formulated as a monomer of a peptide sequence, suchas containing a single individual peptide sequence, or it may beformulated as a multimer of a peptide sequence, i.e containing two ormore individual peptide sequences, wherein said individual peptidesequences may be represented by two or more copies of the same sequenceor by two or more different individual peptide sequences. A multimer mayalso comprises a combination of the full-length sequence and one or morefragments thereof. In one embodiment a compound may contain two aminoacid sequences, such compound is defined herein as dimer, in anotherembodiment a compound may contain more then two amino acid sequences,such for example three, four or more sequences. The present inventionpreferably relates to compounds containing two or four peptide sequencesof the invention. However, compounds containing 3, 5, 6, 7, 8 or moresequences are also in the scope of the invention.

The compounds may be formulated as dimers or multimers comprising morethen two copies of individual peptide fragments which may have theidentical amino acid sequences or different amino acid sequences. Oneexample of such compound may be a dimeric compound containing SEQ ID NO:1 and SEQ ID NO: 2 or a dimeric compound containing SEQ ID NO: 1 and SEQID NO: 3. Any other combinations of the sequences of the invention maybe made depending on different embodiments. The sequences may beconnected to each other via peptide bond, or connected to each otherthrough a linker molecule or grouping.

As already mentioned above, a compound of the invention may contain twoor more copies of a single sequence, such as for example two copies ofany of the sequences selected from SEQ ID NOs: 1-85, wherein said twosequences may be connected to each other via a linker molecule orgrouping. A compound wherein the sequences are connected via a linkergrouping is preferred. One example of such linking grouping may be anachiral di-, tri- or tetracarboxylic acid. Suitable achiral di-, tri- ortetracarboxylic acids and a method of production such a compound (aligand presentation assembly method (LPA)) are described in WO0018791and WO2005014623. Another example of a possible linker may be the aminoacid lysine. Individual peptide sequences may be attached to a coremolecule such as lysine forming thereby a dendritic multimer (dendrimer)of an individual peptide sequence(s). Production of dendrimers is alsowell known in the art (PCT/US90/02039, Lu et al., (1991) Mol. Immunol.28:623-630; Defoort et al., (1992) Int J Pept Prot Res. 40:214-221;Drijfhout et al. (1991) Int J Pept Prot Res. 37:27-32), and dedrimersare at present widely used in research and in medical applications. Itis a preferred embodiment of the invention to provide a dendrimericcompound comprising four individual amino acid sequences attached to thelysine core molecule. It is also preferred that at least one of the fourindividual amino acid sequences comprises an amino acid sequence of theformula defined above. It is even more preferred if the all fourindividual amino acid sequences of a dendrimeric compound individuallycomprise an amino acid sequence of the formula defined above.

Multimeric compounds of the invention, such as LPA-dimers orLysin-dendrmers, are preferred compounds of the invention. However,other types of multimeric compounds comprising two or more individualsequences of the invention may be preferred depending on theembodiments.

3. Biological Activity

A peptide sequence of the invention and a compound comprising a sequenceof the invention possess biological activity. The invention preferablyrelates to a biological activity selected from

-   -   capability of stimulating stem cell proliferation and/or        differentiation, for example neural cell precursor cell        proliferation and/or differentiation;    -   capability of stimulating neural cell differentiation, for        example stimulating neurite outgrowth or regeneration of nerves;    -   capability neural plasticity associated with memory and        learning, for example stimulating synaptic efficacy;    -   capability of stimulating of cell survival, for example        inhibiting apotosis,    -   capability of inhibiting inflammation, such as stimulating        anti-inflammatory response;    -   capability of binding to a receptor, for example Sphingosine        1-phosphate receptor Edg-3 (Swiss-prot Ass. number: Q99500), and        modulating activity of said receptor, such as stimulating or        inhibiting signal transduction associated with this receptor;    -   capability of modulating cell motility, such as inhibiting        cellular migration and cancer cell dissemination;    -   capability of regulating cell differentiation, such stimulating        or inhibiting differentiation of endothelial cells.

Thus, according to one embodiment of the invention a peptide sequence ofthe invention is capable of stimulating neuronal cell differentiation.

The term “neuronal differentiation” is understood herein both asdifferentiation of neural precursor cells, or neural stem cells, andfurther differentiation of neural cells, such as for example maturationof neuronal cells. An example of such differentiation may be neuriteoutgrowth from immature neurons, branching of neurites, and also neuronregeneration.

Thus, one preferred embodiment the invention concerns biologicalactivity of a peptide sequence associated with stimulating ofdifferentiation of neural precursor/stem cells or immature neurons, inanother preferred embodiment the invention concern stimulating neuriteoutgrowth from mature neurons, for examples neurons which weretraumatizes but survived and are committed to regenerate damagedprocesses. Accordingly, the invention also concerns a method forstimulating neuronal cell differentiation comprising using a peptidesequence of the invention or a compound comprising said sequence.

Substances with the potential to promote neurite outgrowth as well asstimulate regeneration and/or differentiation of neuronal cells, such ascertain endogenous trophic factors, are prime targets in the search forcompounds that facilitate for example neuronal regeneration and otherforms of neuronal plasticity. To evaluate the potential of the presentcompound, the ability to stimulate the neurite outgrowth relatedsignalling, interfere with cell adhesion, stimulate neurite outgrowth,regeneration of nerves, may be investigated. Compounds of the presentinvention are shown to promote neurite outgrowth and are thereforeconsidered to be good promoters of regeneration of neuronal connections,and thereby of functional recovery after damages as well as promoters ofneuronal function in other conditions where such effect is required.

In the present context “differentiation” is related to the processes ofmaturation of neurons and extension of neurites, which take place afterthe last cell division of said neurons. The compounds of the presentinvention may be capable of stopping neural cell division and initiatingmaturation said cells, such as initiating extension of neurites.Otherwise, “differentiation” is related to initiation of the process ofgenetic, biochemical, morphological and physiological transformation ofneuronal progenitor cells, immature neural cells or embryonic stem cellsleading to formation of cells having functional characteristics ofnormal neuronal cell as such characteristics are defined in the art. Theinvention defines “immature neural cell” as a cell that has at least onefeature of neural cell accepted in the art as a feature characteristicfor the neural cell.

According to the present invention a compound comprising at least one ofthe above peptide sequences is capable of stimulating neurite outgrowth.The invention concerns the neurite outgrowth improvement/stimulationsuch as about 75% improvement/stimulation above the value of neuriteoutgrowth of control/non-stimulated cells, for example 50%, such asabout 150%, for example 100%, such as about 250, for example 200%, suchas about 350%, for example 300%, such as about 450%, for example 400%,such as about 500%.

Estimation of capability of a candidate compound to stimulate neuriteoutgrowth may be done by using any known method or assay for estimationof neurite outgrowth, such as for example as the described in Examplesbelow.

According to the invention a compound has neuritogenic activity both asan insoluble immobile component of cell growth substrate and as asoluble component of cell growth media. In the present context“immobile” means that the compound is bound/attached to a substancewhich is insoluble in water or a water solution and thereby it becomesinsoluble in such solution as well. For medical applications bothinsoluble and soluble compounds are considered by the application,however soluble compounds are preferred. Under “soluble compound” isunderstood a compound, which is soluble in water or a water solution.

One of most preferred embodiments of the invention concerns the activityof the peptide sequences in connection with learning and memory, inparticular, the capability of a peptide sequence to stimulate synapticplasticity, spine formation, synaptic efficacy. Thus, the invention alsoconcerns a method for stimulating memory and/or learning comprisingusing a peptide sequence of the invention and/or compound comprisingsaid sequence. The invention relates to both shortterm memory andlong-term memory.

In another preferred embodiment of the invention a peptide sequence ofthe invention capable of stimulating cell survival, in particularneuronal cell survival. The invention concerns the capability ofstimulating cell survival both due trauma and degenerative disease.Accordingly, the invention relates to a method for stimulating cellsurvival, preferably neuronal cell survival by using a peptide sequenceof the invention and/or compound comprising said sequence.

Substances with the potential to enhance neuronal cells to survive dueto damage as well as inhibit degeneration and/or apoptosis of neuronalcells in trauma and disease, are prime targets in the search forcandidate compounds for new medicine for treatment of neurodegenerativediseases such as for example Alzheimer's or Parkinson's diseases. Toevaluate the potential of the present peptides, the ability to stimulatesurvival related signalling, interfere with apoptosis related cellularreactions, stimulate regeneration of nerves may be investigated.Compounds of the present invention are shown to promote neural cellsurvival and decrease the cell loss and therefore considered to be goodcandidates for promotion of regeneration of neural connections in brainand/or in peripheral neural system, and thereby of functional recoveryafter damages due trauma or disease as well as promoters of neuronalfunction in any other conditions where such effect is required.

In the present context “survival” is related to the processes associatedwith maintenance and/or recovery of cell function after the damage ofthe cell. The compounds of the present invention may be capable ofstopping or attenuating the processes committing the cell to death, suchas inhibiting apoptosis of neural cells initiated by cell damage duetrauma or disease. Otherwise, “survival” is related to inhibition of theprocesses associated with the cell damage leading to cell death andinitiation of the processes of genetic, biochemical, morphological andphysiological transformation or reconstruction of cells, in particularneuronal cells, such as progenitor cells, immature neural cells orembryonic stem cells or mature neural cells having normal functionalcharacteristics defined in the art. The invention defines “immatureneural cell” as a cell that has at least one feature of neural cellaccepted in the art as a feature characteristic for the neural cell.

According to the present invention a compound comprising at least one ofthe above peptide sequences is capable of stimulating neural cellsurvival. The invention concerns the neural cell survival stimulationsuch as about 75% stimulation above the value of survival ofcontrol/non-stimulated cells, for example 50%, such as about 150%, forexample 100%, such as about 250, for example 200%, such as about 350%,for example 300%, such as about 450%, for example 400%, such as about500%.

Estimation of capability of a candidate compound to stimulate neuralcell survival may be done by using any known method or assay forestimation of cell survival, such as for example the ones described inExamples of the present application.

According to the invention a compound has survival promoting activityboth as insoluble and soluble compound. In the present context“insoluble” means that the compound is bound/attached to a substancewhich is insoluble in water or a water solution and thereby the compoundbecomes insoluble in such solution as well. For medical applicationsboth insoluble and soluble compounds are considered by the application,however soluble compounds are preferred. Under “soluble compound” isunderstood a compound, which is soluble in water or a water solution.

In another embodiment the peptide sequence of the invention is alsocapable of inhibiting an inflammatory process, in particular aninflammatory process in the brain.

Inflammation is a defense reaction caused by tissue damage due to amechanical injury or bacterial, virus or other organism infection. Theinflammatory response involves three major stages: first, dilation ofcapillaries to increase blood flow; second, microvascular structuralchanges and escape of plasma proteins from the bloodstream; and third,leukocyte transmigration through endothelium and accumulation at thesite of injury and infection. The inflammatory response begins with arelease of inflammatory mediators. Inflammatory mediators are soluble,diffusible molecules that act locally at the site of tissue damage andinfection, and at more distant sites, influencing consequent events ofthe inflammatory response. Inflammatory mediators can be exogenous, e.g.bacterial products or toxins, or endogenous, which are produced withinthe immune system itself, as well as injured tissue cells, lymphocytes,mast cells and blood proteins.

Neuroinflammation plays a prominent role in the progression ofAlzheimer's disease and may be responsible for degeneration invulnerable regions such as the hippocampus. Neuroinflammation isassociated with elevated levels of extracellular glutamate andpotentially an enhanced stimulation of glutamate N-methyl-D-aspartatereceptors.

Anti-inflammatory is another important biological activity of thepeptide sequence of the invention. Thus, the invention relates toanti-inflammatory peptide, which is capable of serving as an inhibitorof the sustained inflammatory response, in particular in the brain.

The continuous presence of inflammatory mediators, such as for exampleTNF alpha in the body in response to sustained presence of bacterialproducts or even live bacteria locally during days or weeks followingtrauma and/or infection promotes the reactions to inflammation, such as,for example, heat, swelling, and pain. The sustained inflammatoryresponse has been proven to be very harmful to the body. If thebacterial products or live bacteria become spread universally in thebody from their local focus the inflammatory reaction becomesoverwhelming and out of control and leads to sepsis which eventuallyprogress further to severe sepsis and septic shock. Anti-inflammatorypeptides may be used to block or suppress the overwhelming sustainedinflammatory response represented by a massive and harmful cytokinecascade in the blood and vital organs such as lung, liver intestine,brain and kidneys.

In the present context by the term “anti-inflammatory compound” is meanta compound which is capable of at least one of the following activities

-   i) decreasing or inhibiting the gene expression in the immune cells,    preferably monocytes/macrophages in response to bacterial products,    live bacteria or trauma to produce endogenous inflammatory mediators    including receptors for inflammatory mediators and transcription    factors involved in the signal transduction of the inflammatory    mediators, said mediators being preferably selected from the group    comprising cytokines, selected from the group TNFalpha IL-1, IL-6,    G-CSF, GM-CSF, M-CSF. Chemokines selected from the group comprising    IL-8, MCP-1, receptors selected from the group Tissue factor and    IL-2Ralpha,-   ii) decrease or inhibit the production bradykinin by the phase    contact system,-   iii) decrease or inhibit the attractant potential for monocytes,    and/or-   iv) decrease or inhibit the life-time of monocytes, neutrophils and    other immune cells serving as an inducer of apoptosis,-   v) decrease or inhibit vascular endothelial cells to express the    adhesion molecules, said adhesion molecules being preferably    selected from the group comprising PECAM, ICAM-1, E-selectins,    VCAM-1-   vi) decrease or inhibit activation of the contact phase system to    produce bradykinin leading to increased vascular permeability,-   vii) stimulate the synthesis of an anti-inflammatory mediator    selected from the group of IL-10 and IL-12.-   viii) inhibiting complement activation;-   ix) decreasing the risk of neural cell degeneration in the presence    of chronic neuroinflammation, e.g. neurons which express glutamate    N-methyl-D-aspartate receptors.

A peptide sequence of the invention in still another embodiment iscapable of modulating cell proliferation, such as stimulating cellproliferation.

Most of the differentiated cell populations in a vertebrate are notpermanent: the cells are continually dying and being replaced. Newdifferentiated cell can be produced during adult life in either of twoways: (1) they can form by the simple duplication of the existingdifferentiated cells, which divide to give pairs of daughter cells ofthe same type; or (2) they can be generated from stem cells, which arenot terminally differentiated (that is, they are not at the end of apathway of differentiation), can divide without limit (or at least forthe lifetime of the animal), and when such cell divide, each daughterhas a choice; it can either remain a stem cell or it can embark on acourse leading irreversibly to terminal differentiation.

Compounds of the present invention in one preferred embodiment arecapable of stimulating cell division, i.e. stimulating proliferation ofcells, of both differentiated and stem cells.

Compounds capable of stimulating of terminally differentiated cells areof great value in therapy of traumatised tissues of the body. Thepeptide sequences of the invention or compounds comprising thereof mayfor example be used for stimulating proliferation of hepatocytes in caseof damage and/or degenerative disease of the liver, or they can be usedfor stimulating proliferation of endothelial cells, which may be usefulin treatment of conditions requiring neoangiogenesis.

Stimulating of stem cell proliferation is another preferred embodimentof use of the peptide sequences of the invention. In a particularpreferred embodiment the invention relates to stimulating neural cellprecursor proliferation.

Terminally differentiated cells of neural system, in particular neurons,are not capable to proliferate, and therefore compounds that are capableto increase a number of cells in a damaged due trauma or degenerativedisease neural cell population are prime targets in a search of newmedicine for treatment such conditions. Multiple examples of diseasesand pathological conditions where the peptide sequences of the inventionmay be applied are discussed in the below sections of the description ofthe further embodiments of the invention.

Testing new compounds for the potential of stimulating cellproliferation may be done as described in Examples below.

4. Production of Individual Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the DNA sequences encoding the correspondingfull-length protein of peptide origin, using DNAase I according to astandard protocol (Sambrook et al., Molecular cloning: A Laboratorymanual. 2 rd ed., CSHL Press, Cold Spring Harbor, N.Y., 1989). Thepresent invention relates to full-length proteins selected from thegroups of proteins identified above. The DNA encoding the full-lengthproteins of the invention may altematively be fragmented using specificrestriction endonucleases. The fragments of DNA are further purifiedusing standard procedures described in Sambrook et al., Molecularcloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor,N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell. Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983, Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al, eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4-c (Russell et al., 1983, Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-S-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238-023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteins. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

By SAPS peptides may be synthesised either batchwise in a polyethylenevessel equipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A.and Sheppard, R. C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137.) ona fully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tert.-Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality.

When synthesised, individual peptide sequences may then be formulated asmultimers using well-known in the art techniques, for examples dimers ofthe sequences may be obtained by the LPA method described in WO00/18791, denrimeric polymers by the MAP synthesis described inPCT/US90/02039.

5. Antibody

It is an objective of the present invention to provide an antibody,antigen binding fragment or recombinant protein thereof capable ofrecognizing and selectively binding to an epitope comprising the motifof the invention or a sequence selected from SEQ ID NOs:1-85, or afragment of said sequence, preferably the epitope is located on aprotein of the S100 family, for example S100A4 or S100A12

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies(thereby causing an immune response). The term “epitope” is theequivalent to the term “antigenic determinant”. The epitope may comprise3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 aminoacid residues, located in close proximity, such as within a contiguousamino acid sequence, or located in distant parts of the amino acidsequence of an antigen, but due to protein folding have been approachedto each other.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an anti-gen binding region known asa variable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA. 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains ofantibodies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain. The subunit structures and threedimensional configurations ofdifferent classes of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody which includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody”, as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In preferred embodiments, in thecontext of both the therapeutic and screening methods described below,an antibody or fragment thereof is used that is immunospecific for anantigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom anti-body fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising a peptide sequence selected from any of the sequencesidentified herein as SEQ ID NOs: 1-85, or a fragment of said sequences.Thus, in context of the present invention the term “antibodv fragment”is identical to term “antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per anti-body molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab′ fragments held together by        two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

-   -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule. Such single chain antibodies are also referred to as        “single-chain Fv” or “sFv” antibody fragments. Generally, the Fv        polypeptide further comprises a polypeptide linker between the        VH and VL domains that enables the sFv to form the desired        structure for antigen binding. For a review of sFv see Pluckthun        in The Pharmacology of Monoclonal Antibodies 113: 269-315        Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

The invention contemplate both polyclonal and monoclonal antibody,antigen binding fragments and recombinant proteins thereof which arecapable of binding an epitope according to the invention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726, Cold Spring Harbor Pub. (1988), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, NY.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991,Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Biol 222:581-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate anti-bodies containing human specific andrecognizable sequences. See, for review, Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inanti-bodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567); Morrison et al., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain anti-gen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., 1991, In: Methods: ACompanion to Methods in Enzymology, 2:97; Bird et al., 1988, Science242:423-426; U.S. Pat. No. 4,946,778; and Pack, et al., 1993,BioTechnology 11:1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the eitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising the epitope(s) described herein, is one of the preferredembodiments of the invention.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard method inthe art for producing polyclonal and monoclonal antibodies using naturalor recombinant fragments of human S100 protein, such as S100A4 orS100A12, said fragment comprising a structural motif of the invention,for example comprising a sequence selected from SEQ ID NOs: 1-85, suchas for example a sequences selected from SEQ ID NOs: 1-5, as an antigen.Such antibodies may be also generated using variants, homologues orfragments of peptide sequences of SEQ ID NOs:1-85 said variants,homologues and fragments are immunogenic peptide sequences which meetthe following criteria:

(i) being a contiguous amino acid sequence of at least 6 amino acids;(ii) comprising the motif of the invention.

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of animmunogenic fragment described above.

The invention relates both to antibodies as above, which are capable ofmodulating, such as enhancing or attenuating, biological function ofS100A4 or S100A12, or inhibiting this function. Preferred biologicalfunctions of S100A4 and S100A12 in the present context may be acapability of stimulating cell proliferation, cell differentiation orcell survival, promoting nerve regeneration, inhibiting cellmigration/dissemination, promoting morphological and functionalplasticity, e.g. enhancing synaptic plasticity.

6. Pharmaceutical Composition

The invention also relates to a pharmaceutical composition comprisingone or more of the compounds defined above, wherein the compound iscapable of stimulating neurite outgrowth and/or neural celldifferentiation, survival of neural cells and/or stimulating learningand/or memory. Thus, the invention concerns a pharmaceutical compositioncapable of stimulating differentiation of neuronal cells and/orstimulating regeneration of neuronal cells, and/or stimulating neuronalplasticity in connection with learning and memory, and/or stimulatingsurvival of neural cells.

In the present context the term “pharmaceutical composition” is usedsynonymously with the term “medicament”.

In a composition the peptide sequences may be formulated as comprisingisolated individual peptide fragments or multimers or dimers thereof asdiscussed above.

The pharmaceutical composition may have the described above effects oncells in vitro or in vivo, wherein the composition is administered to asubject.

The medicament of the invention comprises an effective amount of one ormore of the compounds as defined above, or a composition as definedabove in combination with the pharmaceutically acceptable additives.Such medicament may suitably be formulated for oral, percutaneous,intramuscular, intravenous, intracranial, intrathecal,intracerebroventricular, intranasal or pulmonal administration.

Strategies in formulation development of medicaments and compositionsbased on the compounds of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude polyalkylene glycols or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient(s) in the range offrom 0.5% to 10%, preferably 1-2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and generally contain 10-95% of theactive ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaladministration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide compound) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic acid, oxalic acid, tartaric acid,mandelic acid, and the like. Salts formed with the free carboxyl groupmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred μg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 μg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. It is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecornpounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

As discussed above, the present invention relates to treatment ofindividuals for inducing differentiation, stimulating regeneration,plasticity and survival of neural cells in vitro or in vivo, saidtreatment involving administering an effective amount of one or morecompounds as defined above.

Another strategy for administration is to implant or inject cellscapable of expressing and secreting the compound in question. Therebythe compound may be produced at the location where it is going to act.

7. Treatment

In a further aspect, the present invention relates to said peptides,fragments, or variants thereof for use in the induction ofdifferentiation and/or stimulation of regeneration, plasticity and/orsurvival of neural cells. The use is for the treatment for preventingdiseases and conditions of the central and peripheral nervous system,and of the muscles or of various tissues and organs.

Treatment by the use of the compounds/compositions according to theinvention is in one embodiment useful for inducing differentiation,modulating proliferation, stimulate regeneration, neuronal plasticityand survival of cells being implanted or transplanted. This isparticularly useful when using compounds having a long term effect.

Thus, the treatment comprises treatment and/or prophylaxis of cell deathin relation to diseases or conditions of the central and peripheralnervous system, such as postoperative nerve damage, traumatic nervedamage, e.g. resulting from spinal cord injury, impaired myelination ofnerve fibers, postischaemic damage, e.g. resulting from a stroke,multiinfarct dementia, multiple sclerosis, nerve degeneration associatedwith diabetes mellitus, neuro-muscular degeneration, schizophrenia,Alzheimer's disease, Parkinson's disease, or Huntington's disease.

Also, in relation to diseases or conditions of the muscles includingconditions with impaired function of neuro-muscular connections, such asgenetic or traumatic atrophic muscle disorders; or for the treatment ofdiseases or conditions of various or gans, such as degenerativeconditions of the gonads, of the pancreas, such as diabetes mellitustype I and II, of the kidney, such as nephrosis the compounds accordingto the invention may be used for inducing differentiation, modulatingproliferation, stimulate regeneration, neuronal plasticity and survival,i.e. stimulating survival.

In yet a further embodiment the use of the compound and/orpharmaceutical composition is for the stimulation of the ability tolearn and/or of the short and/or long term memory.

In particular the compound and/or pharmaceutical composition of theinvention may be used in the treatment of clinical conditions, such aspsychoses, such as senile and presenile organic psychotic conditions,alcoholic psychoses, drug psychoses, transient organic psychoticconditions, Alzheimer's disease, cerebral lipidoses, epilepsy, generalparesis [syphilis], hepatolenticular degeneration, Huntington's chorea,Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of thebrain, syphilis, Schizophrenic disorders, affective psychoses, neuroticdisorders, personality disorders, including character neurosis,nonpsychotic personality disorder associated with organic brainsyndromes, paranoid personality disorder, fanatic personality, paranoidpersonality (disorder), paranoid traits, sexual deviations anddisorders, mental retardation, disease in the nerve system and senseorgans, cognitive anomalies, inflammatory disease of the central nervoussystem, such as meningitis, encephalitis, Cerebral degenerations such asAlzheimer's disease, Pick's disease, senile degeneration of brain,communicating hydrocephalus, obstructive hydrocephalus, Parkinson'sdisease including other extra pyramidal disease and abnormal movementdisorders, spinocerebellar disease, cerebellar ataxia, Marie's,Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellardegeneration, such as spinal muscular atrophy, familial, juvenile, adultspinal muscular atrophy, motor neuron disease, amyotrophic lateralsclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbarpalsy, primary lateral sclerosis, other anterior horn cell diseases,anterior horn cell disease, unspecified, other diseases of spinal cord,syringomyelia and syringobulbia, vascular myelopathies, acute infarctionof spinal cord (embolic) (nonembolic), arterial thrombosis of spinalcord, edema of spinal cord, subacute necrotic myelopathy, subacutecombined degeneration of spinal cord in diseases classified elsewhere,myelopathy, drug-induced, radiationinduced myelitis, disorders of theautonomic nervous system, disorders of peripheral autonomic,sympathetic, parasympathetic, or vegetative system, familialdysautonomia [Riley-Day syndrome], idiopathic peripheral autonomicneuropathy, carotid sinus syncope or syndrome, cervical sympatheticdystrophy or paralysis; peripheral autonomic neuropathy in disordersclassified elsewhere, amyloidosis, diseases of the peripheral nervesystem, brachial plexus lesions, cervical rib syndrome, costoclavicularsyndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachialneuritis or radiculitis, including in newborn. Inflammatory and toxicneuropathy, including acute infective polyneuritis, Guillain-Barresyndrome, Postinfectious polyneuritis, polyneuropathy in collagenvascular disease. Pain syndrome (such as non-opoid pain, neuropaticpain, or in pain related to other disorders, e.g. diabetes or HIV),encephalitis, drug/alcohol abuse, anxiety, perioperative ischemia,psychoses, such as senile and presenile organic psychotic conditions,alcoholic psychoses, drug psychoses, transient organic psychoticconditions, disorders affecting multiple structures of eye, purulentendophthalmitis, diseases of the ear and mastoid process, abnormality oforgans and soft tissues in newborn, including in the nerve system, bothacute dysfunction and chronic dysfunction (e.g. deficit in cognitionmood social functioning) after injury (peripheral and centrally),complications of the administration of anesthetic or other sedation inlabor and delivery, diseases in the skin including infection,insufficient circulation problem, injuries, including after surgery,crushing injury, burns. Atrophic dermatitis, psoriasis, infection caseddisorders, injuries to nerves and spinal cord, including division ofnerve, lesion in continuity (with or without open wound), traumaticneuroma (with or without open wound), traumatic transient paralysis(with or without open wound), accidental puncture or laceration duringmedical procedure, injury to optic nerve and pathways, optic nerveinjury, second cranial nerve, injury to optic chiasm, injury to opticpathways, injury to visual cortex, unspecified blindness, injury toother cranial nerve(s), injury to other and unspecified nerves,poisoning by drugs, medicinal and biological substances, genetic ortraumatic atrophic muscle disorders; or for the treatment of diseases orconditions of various organs, such as degenerative conditions of thegonads, of the pancreas, such as diabetes mellitus type I and II, of thekidney, such as nephrosis, metabolic disorders such as obscenity lipiddisorders (e.g. hypercholestorolamia, artherslerosis), disorders ofendocrine glands, pituitary gland tumor, disorders of amino acidtransport and metabolism, disorders of purine and pyrimidine metabolismand gout, bone disorders, such as fracture, osteoporosis, osteoarthritis (OA), stem cell protection or maturation in vivo or in vitro,neurogenesis.

In another embodiment the compound and/or pharmaceutical composition ofthe invention may be used in the treatment of neoplasms such asmalignant neoplasms, benign neoplasms, carcinoma in situ and neoplasmsof uncertain behavior, more specifically cancer in breast, thyroidal,pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ(incl. but not limited to CML and AML), muscles (sarcoma). Cancers withdysfunction and/or over- or under-expression of specific receptorsand/or expression of mutated receptors or associated with solublereceptors, such as but not limited to Erb-receptors and FGF-receptors.

Inflammation of the brain is often consequence of infection, autoimmuneprocesses, toxins, and other conditions. Viral infections are arelatively frequent cause of this condition. Encephalitis may occur asprimary or secondary manifestation of TOGAVIRIDAE INFECTIONS;HERPESVIRIDAE INFECTIONS; ADENOVIRIDAE INFECTIONS; FLAVIVIRIDAEINFECTIONS; BUNYAVIRIDAE INFECTIONS; PICORNAVIRIDAE INFECTIONS;PARAMYXOVIRIDAE INFECTIONS; ORTHOMYXOVIRIDAE INFECTIONS; RETROVIRIDAEINFECTIONS; and ARENAVIRIDAE INFECTIONS.

Accordingly, a peptide, compound or a pharmaceutical composition of theinvention may be used for treatment inflammation in the brain associatedwith a viral infection.

A large body of clinical and experimental data indicate that complementactivation is an important mechanism for neuronal and glial injury inGuillain-Barré syndromes. Inhibition of complement activation thereforemight be expected to limit the progression of the disease (Halstead etal. (2005) Annals of Neurology 58:203-21).

Thus, in another embodiment, a peptide sequence, a compound andpharmaceutical composition may be used for treatment of Guillain-Barrésyndrome, its variant forms, such as Miller Fisher syndrome, and othercomplement dependent neuromuscular disorders.

Peptide sequences, compounds and pharmaceutical composition may also beused for treatment children with autism.

Autism is a brain disorder that begins in early childhood and persiststhroughout adulthood; affects three crucial areas of development:communication, social interaction, and creative or imaginative play. Itis estimated to afflict between 2 and 5 of every 1000 children and isfour times more likely to strike boys than girls. Children with autismhave difficulties in social interaction and communication and may showrepetitive behaviour and have unusual attachments to objects orroutines.

In recent years, there have been scientific hints of immune systemirregularities in children with autism.

Thus, a peptide sequence, compound or a composition comprising thereofmay advantageously be used for treatment inflammation, in particularinflammation of the brain.

A further aspect of the invention is a process of producing apharmaceutical composition, comprising mixing an effective amount of oneor more of the compounds of the invention, or a pharmaceuticalcomposition according to the invention with one or more pharmaceuticallyacceptable additives or carriers, and administer an effective amount ofat least one of said compound, or said pharmaceutical composition to asubject.

In one embodiment of the process as mentioned above, the compounds areused in combination with a prosthetic device, wherein the device is aprosthetic nerve guide. Thus, in a further aspect, the present inventionrelates to a prosthetic nerve guide, characterised in that it comprisesone or more of the compounds or the pharmaceutical composition asdefined above. Nerve guides are known in the art.

Another aspect of the invention relates to the use of a compound asdefined above. In particular the use of a compound according to theinvention is for the production of a pharmaceutical composition. Thepharmaceutical composition is preferably for the treatment orprophylaxis of any of the diseases and conditions mentioned above.

In yet a further aspect the invention relates to a method of treating adisease or condition as discussed above by administering a compound asdefined herein.

EXAMPLES Peptides

The sequences of S100A4 and S100A12 proteins were divided into sixregions each, and the tetrameric peptides representing individualregions were generated. The peptides were termed hekatones (H) 1 to 6(FIG. 1). Thereafter, all twelve peptides were tested for theirneuritogenic activity in primary neurons.

S4-3 (A4 H3) KELLTRELPSFLGKRT SEQ ID NO: 2 S4-4 (A4 H4) DEAAFQKLMSNLDSEQ ID NO: 4 S4-6 (A4 H6) NEFFEGFPDKQPRKK SEQ ID NO: 1 S12-1 (A12 H1)TKLEEHLEGIVNIF SEQ ID NO: 60 S12-3 (A12 H3) KQLLTKELANTIKNIK SEQ ID NO:3 S12-4 (A12 H4) DKAVIDEIFQGLD SEQ ID NO: 33 S12-6 (A12 H6) HYHTHKE SEQID NO: 75

1. Primary Neurons: Determination of Neurite Outgrowth

Dissociated hippocampal and cerebellar (CGN) neurons were isolated fromWistar rat embryos at embryonic day 19 or newborn rats as previouslydescribed by Rønn et al. (1999). Briefly, hippocampus was isolated fromthe brain in ice cold modified Krebs Ringer solution, cleared of bloodvessels, roughly homogenised by chopping and then trypsinised. Thedissociated cells were washed in the presence of DNAse 1 and soybeantrypsin inhibitor. Postnatal hippocampal neurons were plated at adensity of 10,000 cells/cm² on uncoated 8-well permanox Lab-Tek chamberslides in Neurobasal medium supplemented with 0.4% (w/v) bovine serumalbumin (BSA; Sigma-Aldrich), 2% (v/v) B27 Neurobasal supplement, 1%(v/v) glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin and 2% 1 MHEPES (all from Gibco, BRL). After 24 hours, the neurons were fixed with4% (v/v) formaldehyde for 20 minutes and thereafter immunostained usingprimary rabbit antibodies against GAP-43 and Alexa Fluor secondary goatanti-rabbit Ig antibodies. Images of at least 200 neurons for each groupin each individual experiment were obtained systematically by computerassisted fluorescence microscopy as previously described (Rønn et al.,2000). Briefly, a Nikon Diaphot inverted microscope with a Nikon Plan20× objective (Nikon, Tokyo, Japan) coupled to a video camera (GrundigElectronics, Germany) was used for recordings. A software package Primadeveloped at the Protein Laboratory (Copenhagen, Denmark) was used tomake a stereologically based determination of neurite length (Rønn etal., 2000).

Results

Neuronal cell cultures were treated with various concentrations of apeptide for 24 hours, and the average neurite length was evaluated asdescribed above. It was found that the peptides A4H3, A12H3 and A4H6strongly induced neurite outgrowth from both cerebellar and hippocampalprimary neurons, whereas other peptides derived from the S100A4 andS100A12 proteins had no effect (FIG. 2). Thus peptide segments number 3and number 6 from various members of the S100 protein family mayrepresent potent pharmacological tools to induce neuronaldifferentiation and axon regeneration.

The H3 and H6 peptides derived from S100A4 were further characterized invitro, and are henceforth referred to as H3 and H6. First, truncatedversions of the peptides were tested for their ability to induce neuriteoutgrowth from primary hippocampal neurons (FIG. 3). Non-truncatedversions were used as controls. It was found that only the C-terminalpart of the H3 peptide was crucial for the induction of neuriteoutgrowth, whereas for the H6 peptides truncation of just two aminoacids from the N-terminal resulted in the loss of neuritogenic activity.Based on truncation experiments, a minimal sequence required for theinduction of neurite outgrowth was identified for each peptide(underlined on top of each truncation graphs). These sequences weresubsequently used for the alascan experiments, where individual aminoacids were substituted by alanins in order to identify residues crucialfor the neurotigenic activities of H3 and H6. We have found four aminoacids in each peptide sequence, which were important for the inductionof neurite outgrowth of H3 and H6 (underlined on top of respectivealascan graphs).

2. Activation of Phosphorylated Akt, CREB, and ERK

Primary hippocampal neurons cultured for 6 hours were stimulated with H3or H6 for 15 minutes. Thereafter the phosphorylation level of the threeintracellular messengers involved in neurite outgrowth and survival,Akt, CREB, and ERK, was determined using the PAGE method.

Results

It was found that both peptides significantly increased phosphorylationof all three messengers (FIG. 4).

3. Neuronal Survival.

Primary cerebellar neurons were cultured for 7 days in high potassiummedium, KCl concentration=40 mM. The primary cerebellar neurons wereinduced to undergo apoptosis by lowering of the KCl concentration to 5mM. Both S100A4-derived peptides H3 and H6 partially rescued cerebellarneurons and increased the neuronal survival by 30-40% (FIG. 5).

Since Ca²⁺ has been demonstrated to play a crucial role in the processesof neurite outgrowth and survival, and S100A4 is known to increase[Ca²⁺]_(i) in primary neurons, we loaded cultured hippocampal neuronswith a ratiometric Ca²⁺-sensitive dye fura-2 and tested whether theS100A4-derived peptides affected [Ca²⁺]_(i). It was found thatapplication of H6, but not H3 significantly increased the level ofintracellular Ca²⁺ (FIG. 6).

4. Kainic Acid-Induced Toxicity

Male C57BL/6J mice were injected with 10 mg/kg H3 or H6 daily startingfrom day-2 prior to kainic acid treatment. At day 0 the animals wereinjected 30 mg/kg KA in order to induce severe seizures, or 20 mg/kg KAin order to induce mild seizures and prevent excessive mortality.Latency and seizure severity were scored for 2 h postinjection by usinga numerical scale of 0 to 6: 0—immobility; 1—facial automatism; 2—headnodding; 3—forelimb clonus; 4—rearing; 5—generalized convulsions;6—death. Control animals received s.c injection of vehicle.

Results

H3 and H6 protect the brain from the kainic acid (KA)-induced toxicity(FIG. 7). Both H3 and H6 decreased the amount of seizures induced by 20mg/kg KA. Moreover, the peptides shifted the profile of seizures inducedby 30 mg/kg KA towards less severe seizures types and decreased themortality by 30-50%.

5. Functional Recovery after Sciatic Nerve Crush

Nerve crush of sciatic nerve was performed as follows. Before surgery,animals were anesthetized with subcutaneous injection of aFenthanil/Droperidol/Medazolam (0.8 ml per 100 g). The right hind limbwas shaved and swabbed with antiseptic solution. A longitudinalcutaneous incision was made in the back of the thigh. Dissection wascarried out along a plane separating the hamstring and gluteal musclesto expose the sciatic nerve. Careful dissection was performed to isolatethe sciatic nerve from the surrounding connective tissue over a lengthof 2 to 2.5 cm. The sciatic nerve was unilaterally crushed twice in thesame place (0.5 cm upper trifurcation) for a 30 sec using a lockingsurgical needle holder. The place of the crush was labelled with 5-0suture material attached to the epineurium. The wound was closed with5-0 suture material and rats were allowed to recover. The opposite legand sciatic nerve were not operated and served as a control. The walkingtrack test was started preoperatively and continuing every second daythroughout the duration of the study atsrting at day 6 after the nervecrush. Both hind paws were pressed against a regular stamppad containingwater-soluble nontoxic ink, and the rat was allowed to walk freelythrough a 15×40 cm corridor, toward a darkened box. The bottom of thecorridor was lined with paper, upon which the hind pawprints of the ratwere recorded. Each rat was allowed to walk 1-3 times per test to obtainthe best possible representative tracks. Several prints of each footwere inspected, and the best corresponding pair of prints fromexperimental (E) and normal (N) legs were measured to the nearestmillimeter, using a caliper. Three footprint parameters were measured:distance from the heel to the third toe, print length (PL), the distancebetween the first and fifth toe, toe spread (TS), and distance betweenthe second and fourth toe, the intermediary toe spread (IT). The threeparameters were combined to estimate the Sciatic Functional Index (SFI)as described by Bain et al., 1988. The SFI varies between 0 (foruninjured) and about (−)100 (for maximally impaired gait).

Results

Wistar rats have been subjected to a sciatic nerve crush at day 0.Thereafter, rats were divided into three groups, each group receivingdaily injections of either vehicle (Veh), or the H3 and H6 peptides at adose of 10 mg/kg for 18 days. Every second day, the Sciatic FunctionIndex (SFI) was evaluated in all groups based on the analysis of walkingtracks of individual animals (SFI=−100 corresponding to a cornplete limbparalysis, SFI=0 corresponding to a normal limb function). It has beenfound that the H3 peptide strongly promoted the recovery of the motorfunction of the injured limb.

6. Traumatic Brain Injury

A focal brain injury on the right fronto-parietal cortex was made byapplying a piece of dry-ice (−78° C.) directly onto the skull for 30seconds in mice and 60 seconds in rats, as previously described indetails (Penkowa et al., 2003).

Tissue Processing

Histochemistry and immunohistochemistry were performed on sections cutfrom organs taken from fixated animals. Rats and mice were deeplyanesthetized with Brietal and flushed by cardiac perfusion with salinecontaining heparine (0.9% NaCl, 3 ml/L 5000IU heparine) for 2 minutesfollowed by fixation with Zamboni's fixative (pH 7.4) for 4-8 minutesdepending on the size of the animal. For immunohistochemicalinvestigation, brain were dissected and postfixed in Zamboni's for 2-3hours, dehydraded in graded alcohol followed by xylol and subsequentlyembedded in paraffin before being cut in 3 μm frontal sectionsthroughout the entire area of the cryo lesion. For heat-induced epitoperetrieval, the sections were boiled in citrate buffer (pH 6 or 9) in amicrowave oven for 10 minutes followed by incubation in 10% goat serum(In Vitro, Fredensborg, DK) or donkey serum (code BP 005.1; The BindingSite, Birmingham, UK) in tris buffered saline (TBS)/Nonidet P-40 (0.01%)for 30 minutes at room temperature. Sections with mouse tissue used forincubation with monoclonal mouse-derived antibodies were also incubatedwith Blocking Solutions A+B from the HistoMouse-SP kit to quenchendogenous mouse IgG (Zymed, Calif., USA).

Immunohistochemistry

Following treatment as described above, sections were incubatedovernight at 5° C. with one of the following primary antibodies:polyclonal rabbit anti-cow glial fibrillary acidic protein (GFAP) 1:250(a marker for astrocytes; DakoCytomation); monoclonal mouse anti-ratproliferating cellular nuclear antigen (PCNA) 1:10 (markingproliferating cell nuclear antigen/cell proliferation; DakoCytomation).The primary antibody was detected with biotynilated secondaryantibodies, incubated with streptavidinbiotin-peroxidase complex for 30min.

In Situ Detection of DNA Fragmentation (TUNEL)

Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate (dUTP)-biotin nick end labelling (TUNEL) was performedusing the Fragment End Labeling (FragEL™) Detection Kit (Calbiochem,USA, code QIA33). The FragEL kit contains all the materials used belowand each step was performed according to the manufacturer'srecommendations. The tissue was processed and rehydrated as mentionedabove, and sections were incubated with 20 □g/ml proteinase K for 20 minto strip off nuclear proteins. After immersion in equilibration bufferfor 20 min, sections were incubated with TdT and biotin-labeleddeoxynucleotids (dNTP-biotin) in a humified chamber at 37° C. for 1.5hr. This was followed by wash buffer and the stop solution for 5 min atroom temperature to stop the reaction. After washing in TBS andincubation in blocking buffer for 10 min, the sections were incubatedwith Peroxidase-Streptavidin for 30 min and afterwards, DAB was used aschromogen. The sections were counterstained with methyl-green. Negativecontrol sections were treated similarly but incubated in the absence ofTdT enzyme or dNTP-biotin or Peroxidase-Streptavidin. We also comparedour sections with positive control slides provided in the FragELDetection Kit. Furthermore, TUNEL was compared with other stainings forapoptosis (activated caspase-3, cytochrome-c and TNF□-R1). In addition,the morphologic criteria for apoptosis, i.e. cell shrinkage, formationof apoptotic bodies, membrane blebbing, no loss of cellular integrity,compaction of chromatin into uniformly dense masses, were evaluated.

Cell Counts and Statistical Analysis

In addition to morphological evaluation, quantification (cellularcounts) of some of the variables, i.e. GFAP-positive cells, the area ofGFAP-immunoreactivity, % of TUNEL-positive cells, % of proliferatingcells, were analyzed. The analysis was carried out from the penumbraarea of 3 μm brain sections for statistical analysis of the results. Foreach parameter analysed, cell counts were performed in at least 2sections from each brain and from at least 5 animals from each group anda mean value was calculated. To this end, positively stained cells weredefined as cells with staining of the soma. However, in the case ofapoptotic markers (like TUNEL) positively stained cells were defined ascells with nuclear staining. The cell counts were performed by the sameinvestigator, who was blinded to animal identity and the differenttreatments. Cells were counted at the border of the cortical lesion (therim between lesioned and unlesioned brain tissue), where inflammation isprominent. Results obtained from sectioned brains were analysed withtwo-way ANOVA where applicable (as judged by Levine's test) or theMann-Whitney test. Results obtained from cell cultures were analyzedwith student's t-test.

Results

(FIGS. 9, 10, 11, 12, 13). Treatment with the peptides (10mg/kg/administration) was started one day before the lesion andcontinued on the next day and the third day after the lesion. Theanimals were perfused on the fourth day after the lesion. It was foundthat both the S4-3 and S4-6 peptides protected cells in the cortex fromcell death induced by cryolesion to undergo apoptosis. Treatment witheach of the two peptides induced astrogliosis as reflected by anincrease of the number of GFAP-positive cells and in the area ofGFAP-immunoreactivity. On the other hand, the A4H4 peptide stronglyinduced proliferation of neural progenitor cells.

CONCLUSION

The peptides S4-3 (A4H3), S4-6 (A4H6) and S12-6 (A12H6) are potentstimulators of neurite outgrowth in vitro.

The peptides S4-3 (A4H3) and S4-6 (A4H6) are neuroprotective in vivo.

The peptide S4-4 (A4H4) has the potential to induce neurogenesis inanimals after brain injury.

1. A peptide comprising at most 30 contiguous amino acid residuescomprising an amino acid motif of the formula:x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4), wherein x^(p1) and x^(p2) arehydrophobic amino acid residues, at least one of (x1), (x2) or (x3) isan amino acid residue selected from E, D, K, R, Q, N, S or T and (x4) isan amino acid residue selected from E, D, K, R, Q, N, S or T or ahydrophobic amino acid residue.
 2. The peptide according to claim 1,wherein the motif comprises a threeamino acid residues sequence whereintwo amino acid residues are hydrophobic residues contiguous to eachother and the third amino acid residue is a residue selected from E, D,K, R, Q, N, S or T.
 3. The peptide according to claim 2, wherein thesequence comprises residues (x3), x^(p2) and (x4), wherein (x3) is anamino acid residue selected from E, D, K, R, Q, N, S or T and (x4) is ahydrophobic amino acid residue, or wherein (x4) is an amino acid residueselected from E, D, K, R, Q, N, S or T and (x3) residue is a hydrophobicamino acid residue.
 4. The peptide according to claim 1, wherein thepeptide comprises 6 to 25 contiguous amino acid residues.
 5. The peptideaccording to claim 1, wherein the peptide comprises 6 to 15 contiguousamino acid residues.
 6. The peptide according to claim 1, wherein thepeptide is capable of stimulating cell survival, cell proliferationand/or cell differentiation.
 7. The peptide according to claim 6,wherein the cell is a cell of the neural system.
 8. The peptideaccording to claim 6, wherein the cell is a neuronal cell.
 9. Thepeptide according to claim 1, wherein the peptide is capable ofstimulating neural plasticity associated with memory and learning. 10.The peptide according to claim 1, wherein the peptide comprises an aminoacid sequence selected from SEQ ID NOs:1-85, or a fragment or a variantof said sequence.
 11. The peptide according to claim 10, wherein thefragment is an amino acid sequence comprising at least 5 amino acidresidues comprising two contiguous hydrophobic amino acid residues and aresidue selected from K, R, D, E, N, Q, S or T which follows or precedessaid hydrophobic residues.
 12. The peptide according to claim 10,wherein the variant is an amino acid sequence of at least 6 amino acidresidues having at least 50% sequence similarity with a sequenceselected from the sequences of SEQ ID NOs: 1-85.
 13. The peptideaccording to claim 10, wherein the peptide is derived from the family ofS100 proteins.
 14. The peptide according to claim 13, wherein thepeptide has a sequence selected from SEQ ID NOs:1-85.
 15. The peptideaccording to claim 13, wherein the peptide has a sequence selected fromSEQ ID NOs: 1-5.
 16. The peptide according to claim 13, wherein thepeptide has a sequence selected from SEQ ID NOs: 51-68.
 17. The peptideaccording to claim 13, wherein the peptide has a sequence selected fromSEQ ID NOs:6-22.
 18. The peptide according to claim 13, wherein thepeptide has a sequence selected from SEQ ID NOs:23-42.
 19. The peptideaccording to claim 12, wherein the peptide has a sequence selected fromSEQ ID NOs:43-50.
 20. The peptide according to claim 12, wherein thepeptide has a sequence selected from SEQ ID NOs:52-68.
 21. The peptideaccording to claim 13, wherein the peptide has a sequence selected fromSEQ ID NOs:69-85.
 22. A compound comprising a peptide of claim
 1. 23.The compound according to claim 22, wherein the peptide is formulated asmonomer consisting of the single copy of an individual peptide sequence.24. The compound according to claim 22, wherein the peptide isformulated as a multimer consisting of two or more copies of anindividual peptide sequence, such as a dimer or tetramer of anindividual peptide sequence.
 25. The compound of claim 24, wherein themultimer is a dendrimer.
 26. (canceled)
 27. A pharmaceutical compositioncomprising a peptide according to claim
 1. 28. A method of stimulatingneural cell survival, differentiation, proliferation and/or plasticityassociated with memory and learning comprising: using a peptide,compound or pharmaceutical composition comprising an amino acid motif ofthe formula: x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4), wherein x^(p1) andx^(p2) are hydrophobic amino acid residues, at least one of (x1), (x2)or (x3) is an amino acid residue selected from E, D, K, R, Q, N, S or Tand (x4) is an amino acid residue selected from E, D, K, R, Q, N, S or Tor a hydrophobic amino acid residue.
 29. A method of treatment of acondition or disease wherein stimulating neural cell survival,differentiation, proliferation and/or plasticity, such as plasticityassociated with learning and memory is beneficial for treatment,comprising: administering an effective amount of a peptide, compound orpharmaceutical composition comprising an amino acid motif of theformula: x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4), wherein x^(p1) and x^(p2)are hydrophobic amino acid residues, at least one of (x1), (x2) or (x3)is an amino acid residue selected from E, D, K, R, Q, N, S or T and (x4)is an amino acid residue selected from E, D, K, R, Q, N, S or T or ahydrophobic amino acid residue.
 30. The method according to claim 29,wherein the condition or disease is a condition or disease of thecentral and peripheral nervous system.
 31. The method according to claim30, wherein the condition or disease is selected from postoperativenerve damage, traumatic nerve damage, impaired myelination of nervefibers, postischaemic damage, such as after stroke, nerve degenerationassociated with diabetes mellitus, disorders affecting the circadianclock or neuromuscular transmission.
 32. The method according to claim29, wherein the condition or disease is selected of conditions ordiseases of the muscles including conditions with impaired function ofneuro-muscular connections, such as after organ transplantation, or suchas genetic or traumatic atrophic muscle disorders.
 33. The methodaccording to claim 29, wherein the condition or disease is an impairedability to learn and/or impaired memory.
 34. The method according toclaim 29, wherein the condition or disease is Parkinson's disease,Alzheimer's disease, Huntington's disease or dementia such asmultiinfarct dementia.
 35. The method according to claim 29, wherein thecondition or disease is a mental disease, such as a disorder of thoughtand/or mood, neuropsychiatric disorders including bipolar (BPD),genetically related unipolar affective disorders, delusional disorders,paraphrenia, paranoid psychosis, schizophrenia, schizotypal disorder,schizoaffective disorder, schizoaffective bipolar and geneticallyrelated unipolar affective disorders, psychogenic psychosis, catatonia,periodic bipolar and genetically related unipolar affective disorders,cycloid psychosis, schizoid personality disorder, paranoid personalitydisorder, bipolar and genetically related unipolar affective disordersrelated affective disorders and subtypes of unipolar affective disorder.36. The method according to claim 29, wherein the disease is priondisease.
 37. The method according to claim 29, wherein the condition ordisease is Guillain-Barré syndrome, its variant form, such as MillerFisher syndrome, or another complement dependent neuromuscular disorder.38. A method of treating cancer, comprising: administering an effectiveamount of a peptide, compound or pharmaceutical composition comprisingan amino acid motif of the formula: x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4),wherein x^(p1) and x^(p2) are hydrophobic amino acid residues, at leastone of (x1), (x2) or (x3) is an amino acid residue selected from E, D,K, R, Q, N, S or T and (x4) is an amino acid residue selected from E, D,K, R, Q, N, S or T or a hydrophobic amino acid residue.
 39. The methodaccording to claim 38, wherein the cancer is any cancer involvingneoangiogenesis.
 40. The method according to claim 38, wherein thecancer is a cancer of neural system.
 41. A method of treating bodydamages due to alcohol consumption, comprising: administering aneffective amount of a peptide, compound or pharmaceutical compositioncomprising an amino acid motif of the formula:x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4), wherein x^(p1) and x^(p2) arehydrophobic amino acid residues, at least one of (x1), (x2) or (x3) isan amino acid residue selected from E, D, K, R, Q, N, S or T, and (x4)is an amino acid residue selected from E, D, K, R, O, N, S or T or ahydrophobic amino acid residue.
 42. A method of treating a condition ordisease which is characterized by the presence of a sustainedinflammatory response, comprising: administering an effective amount ofa peptide, compound or pharmaceutical composition comprising an aminoacid motif of the formula: x^(p1)-(x1)-(x2)-(x3)-x^(p2)-(x4), whereinx^(p1) and x^(p2) are hydrophobic amino acid residues, at least one of(x1), (x2) or (x3) is an amino acid residue selected from E, D, K, R, Q,N, S or T, and (x4) is an amino acid residue selected from E, D, K, R,O, N, S or T or a hydrophobic amino acid residue.
 43. The methodaccording to claim 42, wherein the condition or disease is braininflammation or autoimmune disease.
 44. (canceled)
 45. An antibodycapable of recognizing and binding to an epitope comprising the aminoacid motif according to claim
 1. 46. The antibody according to claim 45,wherein the epitope comprises an amino acid sequence selected from SEQID NOs:1-85, or a fragment or variant of said sequence.
 47. A method oftreating a condition or disease, comprising: administering to anindividual in need an effective amount of an antibody according to claim45.
 48. A pharmaceutical composition comprising compound according toclaim 22.